phasic square wave pulses (0.2 msec, 3 Hz) were delivered through a constant current generator (Grass ... Parada et al. (15) using the 30K antibody. Plasma ...
0013-7227/82/1114-1127S02.0O/O Endocrinology Copyright © 1982 by The Endocrine Society
Vol. I l l , No. 4
Printed in U.S.A.
Tooth Pulp Stimulation Potentiates the Adrenocorticotropin Response to Hemorrhage in Cats* DAVID A. BEREITER, PAUL M. PLOTSKY, AND DONALD S. GANN Section of Surgery, Brown University, Division of Biology and Medicine, Rhode Island Hospital, Providence, Rhode Island 02902
ABSTRACT. Stimulation of the maxillary tooth pulp nerve (TP), a predominantly nociceptive afferent fiber system, was assessed for its effect on peripheral plasma ACTH in chloraloseurethane anesthetized cats. These results were compared to those after a transient 10 ml/kg hemorrhage (H), a submaximal neurogenic stressor for ACTH release, and to H plus TP in combination. TP alone for 3 min had no significant effect on ACTH. However, TP during H greatly potentiated the increase in plasma ACTH concentration compared to that seen after H alone. The TP potentiation of the H-induced rise in ACTH was
P
nary report on this study has been published previously (10).
AIN HAS BEEN included among the group of neurogenic stressors that facilitate the release of ACTH; however, previous investigators either have not measured ACTH directly (1, 2) or have employed noxious stimuli that did not activate nociceptors selectively and that
Materials and Methods Animal preparation
were difficult to quantify (1, 3, 4, 5). To assess the effects
of nociceptor stimulation on ACTH release, we have elected to stimulate a peripheral nerve system [tooth pulp (TP)], thought to convey predominantly nociceptive afferent information, while measuring ACTH. Hemorrhage (H) is a well-known stimulus for the release of ACTH that requires activation of identified peripheral neural receptors (6, 7, 8). The interaction between two distinct populations of peripheral neural receptors on plasma ACTH has not been previously studied, although earlier reports (9) have suggested that H combined with neural stimulation led to lower survival rates in dogs than did H alone. Therefore, a second major aim of this study was to compare directly the effects of nociceptor stimulation on ACTH to the effect of mild H and to the effect of H plus nociceptor stimulation. The results indicate that stimulation of a TP nerve does not activate release of ACTH, but that this stimulation potentiates the response of ACTH to small H. A prelimiReceived February 8, 1982. Address correspondence and requests for reprints to: Donald S. Gann, M.D., Section of Surgery, Division of Biology and Medicine, Rhode Island Hospital, Providence, Rhode Island 02902. * This study was funded by NIH Grants AM-26831 and GM-27946.
not accompanied by altered cardiovascular responsiveness nor by differences in plasma norepinephrine or glucose relative to that seen after H alone. The data indicate that nociceptive and baroreceptor afferents share a common neural substrate for selective facilitation of ACTH release, but do not interact to potentiate several other physiological responses, such as sympathetic efferent activity. Furthermore, under the conditions of these experiments, selective nociceptor activation in the anesthetized cat is not an adequate stimulus for the release of ACTH. (Endocrinology 111: 1127, 1982)
Eighteen adult cats of either sex were deprived of food overnight, but were given free access to water. After initial induction with ketamine-HCl (30 mg/kg, im), anesthesia was maintained with a mixture of a-chloralose-urethane (40/200 mg/kg, iv) administered as a full dose before surgical preparation of the animal with supplemental doses (10/50 mg/kg) given every 2 h throughout the day. An endotracheal tube was inserted, and all animals were placed on a respirator. The paralytic agent gallamine triethiodide (initial dose, 7.5 mg/kg, iv) was given immediately after establishing the current threshold for the jaw-opening reflex to TP stimulation (see below), and supplemental doses (5 mg/kg, iv) were administered hourly thereafter. Catheters were placed in the descending aorta (blood pressure, heart rate monitor), in the femoral artery (hemorrhage), and in the inferior vena cava (blood samples). A brachial vein catheter was used for all infusions. All incisions were infiltrated with 2% xylocaine jelly. Body temperature was maintained at 38.5 C with a heating blanket and rectal temperature probe. Two stainless steel electrodes were inserted into a maxillary canine tooth for TP stimulation. One electrode made contact with the distal pulp after removing the distal crown of the tooth. The second electrode made contact with the pulpal nerves via a lateral burr hole near the gingival border. Monophasic square wave pulses (0.2 msec, 3 Hz) were delivered through a constant current generator (Grass Instruments,
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BEREITER, PLOTSKY, AND GANN
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Springfield, MA). Current amplitude for TP stimulation in each cat was 2x the threshold current for the TP-induced jawopening reflex or a minimum of 100 [xA (current range, 100-300 jiiA). The TP electrodes were then fixed in place with dental acrylic cement.
D H 0 TP • H+TP 600-
Experimental protocol Beginning at least 1.5 h after completion of all surgical procedures, each of 3 stimuli was presented to all 18 cats in a random sequence over a 6-h period. A period of 90 min preceded each additional stimulus presentation. Stimuli consisted of: 1) 10 mg/kg H removed over 1 min, withheld for 2 min, then reinfused over 1 min; 2) TP stimulation as described above for 3 min; or 3) H plus TP, as in 1 and 2. Blood samples (5 ml) were collected into chilled heparinized tubes at —5 and 0 min (prestimulus), +2 min (during stimulation), and +5, +10, and +15 min (post stimulation). Simultaneous fluid replacement with a dextran solution (3 ml) accompanied each blood sample. After centrifugation and removal of plasma for determinations, the red blood cells were resuspended in 0.9% saline and given back to the animal 60 min before the next stimulation period. Animals were killed at the end of each experiment with an anesthetic overdose followed by 2 ml saturated KC1 given intraarterially. Determinations Plasma samples were stored at —20 C for subsequent measurement of hormones determined by RIA. ACTH was measured by the methods of Rees et al. (11) as modified and described previously (12). Plasma cortisol was measured by the method of Ruder et al. (13). Plasma glucagon was measured after acid extraction (14) according to the technique of AguilarParada et al. (15) using the 30K antibody. Plasma catecholamines were measured by high performance liquid chromatography with electrochemical detection (16). Plasma glucose was determined by the glucose oxidase technique (Yellow Springs Instruments, Yellow Springs, OH), and osmolality was measured by freezing point depression (Advanced Instruments, Needham, MA).
Statistical analyses Mean changes from baseline were analyzed by analysis of variance corrected for repeated measures in the same subject (17) with the aid of a NOVA Eclipse computer (Data General, Southboro, MA). Individual comparisons were made by Dunnett's t test (17) after analysis of variance.
Results Plasma ACTH responses to H, TP, and Hplus TP After a mild H, there was a significant (P < 0.01) increase in plasma ACTH concentration that was different from the mean of the 2 control samples (Fig. 1). This was a consistent observation, as 15 of 18 cats demonstrated such an increase in ACTH, whereas only 3 of 18 animals failed to show an increase in ACTH in excess of
400-
O
0.1). Concentrations of peripheral cortisol were elevated for all three experimental groups (~8-9 jug/dl) before stimulation, probably reflecting the elevated ACTH. TP alone did not further elevate cortisol concentrations above control. H alone had little effect on cortisol levels, although at +2 min cortisol concentrations were significantly (P < 0.05) increased above baseline. H plus TP significantly (P < 0.05) increased cortisol above control only at +15 min. The total increase in cortisol above baseline for the H plus TP condition (unweighted cumulative effect for all time points) was significantly different from baseline (P < 0.05) as well as from the H alone condition (P < 0.05) but not different from TP alone.
D
H & TP
• H*TP
Z 30-|
i CO
HI
2010-
a. x 95 pg/ml) that exceeded the interassay coefficient of variation. A general tendency toward in-
-60-
1
2 3
5
10
15
TIME (MIN)
FIG. 2. Heart rate (top) and MAP (bottom) response to H, TP, and H plus TP. Baseline values for heart rate in each group are as follows: H, 238 ± 8 beats per min; TP, 240 ± 6 beats per min; and H plus TP, 236 ± 6 beats/min (mean ± SEM; n = 18). Baseline values for MAP in each group are as follows): H, 119 ± 5 mmHg; TP, 120 ± 5 mm Hg; and H plus TP, 130 ± 4 mm Hg (mean ± SEM; n = 18). *, P < 0.05 vs. baseline; **,P
